Lateral flow strip assay of a gene segment in the COVID-19 virus with combined dual readout mode and preliminary multisite hybrid chain reaction amplification

Anal Methods. 2024 May 23;16(20):3249-3255. doi: 10.1039/d4ay00074a.

Abstract

The past and present scenario of COVID-19 has revealed the necessity of simple point-of-care tests. When combined with the great advantages of amplification, lateral flow assay nucleic acid analysis represents a more sensitive molecular diagnostic technique compared to universal protein analysis. Room temperature operation, an enzyme-free nature, and in situ elongation make hybrid chain reaction amplification (HCR) a good candidate for amplified combined lateral flow assays (LFAs). Since dual modes of detection can not only satisfy different application scenarios, but also reduce the false-negative rate, in this paper, visual and fluorescent detection based on labelling with colloidal gold nanoparticles and fluorescence labelling were incorporated into a HCR integrated with a LFA. The detection assay was finished in 30 minutes. The linear relationship between the signal and the concentration of the characteristic segment in the COVID-19 ORF gene was demonstrated. The obtained detection limits of as low as 10 fM (6.02 × 103 copies per mL) and 1 fM (6.02 × 102 copies per mL), respectively, were comparable with those in the literature. The multi-site HCR amplification integrated with LFA of a 1053 bp nucleic acid chain was also preliminarily studied, and tri-site amplification was found to exhibit higher signal intensity than single-site amplification. This study provides a promising strategy for simple, sensitive, and wide-ranging detection of pathogenic bacteria.

MeSH terms

  • COVID-19 Nucleic Acid Testing / instrumentation
  • COVID-19 Nucleic Acid Testing / methods
  • COVID-19* / diagnosis
  • Humans
  • Limit of Detection
  • Metal Nanoparticles / chemistry
  • Molecular Diagnostic Techniques / methods
  • Nucleic Acid Amplification Techniques* / methods
  • RNA, Viral / analysis
  • RNA, Viral / genetics
  • SARS-CoV-2* / genetics

Substances

  • RNA, Viral